The long-term goal of this project is to identify the molecular, biochemical, and cellular events that control B cell differentiation. Our focus will be on understanding the role of the pre-B cell receptor (pre-BCR). The preBCR controls multiple differentiative events including allelic exclusion, differentiation to pre-BII, clonal expansion, and L chain rearrangement. The hypothesis to be tested is that these functions are differentially sensitive to pre-BCR expression level and that there is a hierarchy of function. Our analysis will center on mice with Ig VH12 H chain transgenes. VH12 pre-B cell selection during differentiation is extreme, providing a unique opportunity to further our understanding of pre-BCR function. Selection favors VH12 pre-B cells with VHCDR3 sequences of 10 amino acids and a Gly in the fourth position, designated 10/G4. 10/G4 pre-B cells survive and differentiate to the B cell stage, whereas the majority (>95%) of non-10/G4 VH12 B cells die at the pre-BI to pre-BII cell transition. To determine the basis for the loss of most non- 10/G4 pre-B cells, we have generated a non-10/G4 transgenic (Tg) mouse line with a VH12 rearrangement encoding an 8 amino acid CDR3 and no Gly (8/GO). The 8/GO pre-BCR induces allelic exclusion, differentiation to pre-BII, and L chain rearrangement, but does not induce pre-B cell clonal expansion or differentiation to a B cell. We hypothesize this deficiency is due to low pre-BCR expression level. Three specific aims are proposed to test this hypothesis and to determine a hierarchy of function. In the first, the level of 8/GO pre-BCR expression on the cell surface and in lipid rafts of ex vivo 8/GO pre-B cells will be determined. In addition, signal transduction and lipid raft translocation will be compared between 8/GO and 10/G4 pre-BCRs. In the second the level of the pre-BCR will be manipulated to determine a hierarchy of pre-BCR function through the use of retroviral bone marrow infection and the establishment of additional non-10/G4 Tg mice. In the final aim whether the basis for the inability of 8/GO Tg mice to generate immature B cells is due to a pre-BCR or BCR deficiency will be determined.